PROJECT SUMMARY/ABSTRACT Alzheimer?s disease (AD) is a devastating, progressive neurodegenerative disease affecting an estimated 11% of Americans over 65 years of age and 33-50% over 85 years of age. The initial pathological sign of AD, the build-up of beta amyloid (A?) plaques in the brain, often precedes impairment of cognition. Although these plaques can be imaged by A? PET brain scans or detected via analysis of A? peptides in the cerebrospinal fluid, these methods are costly, invasive and inconvenient. Recently, with clinical studies indicating that therapeutics may be more effective in treating AD at the earliest stages of the disease and that anti-A? therapy can remove A? from the brain, the need for a simple, inexpensive diagnostic blood test for AD onset and progression is even greater. It has been difficult to develop a blood test based on A?, in part because of the low concentration of A? peptides in blood and the high concentration of other proteins. We have approached this protein design problem by developing methods to link the antigen binding domains (Fabs) of antibodies to beads using flexible and extendible non-peptidyl hinges. The flexibly-linked Fabs on the beads are able to efficiently capture exceptionally low levels of A? (307 attomoles) in blood by binding cooperatively in a two-handed fashion. These Flex-Hinge? Fab beads, combined with MALDI-TOF mass spectrometry, have revealed previously unknown forms of A? including a novel A? biomarker in plasma that acts as a precise qualitative and quantitative proxy for brain A? PET imaging, correlating with the stages of AD. In this Phase I SBIR, we shall improve upon these results in two key ways: (1) instead of using Flex-Hinge? Fab beads, we shall produce Flex-Hinge? Bispecific Antibody beads that we expect to have even greater sensitivity and utility through improved cooperativity and reduced variability, and (2) we shall move to Orbitrap technology, which has greater sensitivity and resolution for A? peptides compared to MALDI. We shall use these Flex-Hinge? Bispecific Antibody beads and Orbitrap technology to examine clinical samples from the Swedish BioFINDER study to confirm the utility of the novel A? biomarker and identify additional A? biomarker(s) in plasma. Phase II studies shall evaluate this assay with larger sample sets. Ultimately, we intend to turn our test into a kit that will be broadly available and become routine for monitoring AD, much as cholesterol testing is used for monitoring cardiovascular disease.